molecules

Article Time-dependent Inhibition of CYP2C8 and CYP2C19 by Hedera helix Extracts, A Traditional Respiratory Herbal Medicine

Shaheed Ur Rehman 1, In Sook Kim 2, Min Sun Choi 2, Seung Hyun Kim 3, Yonghui Zhang 4 and Hye Hyun Yoo 2,*

1 Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan; [email protected] 2 Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Ansan, Gyeonggi-do 15588, Korea; [email protected] (I.S.K.); [email protected] (M.S.C.) 3 College of Pharmacy, Yonsei Institute of Pharmaceutical Science, Yonsei University, Incheon 21983, Korea; [email protected] 4 School of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China; [email protected] * Correspondence: [email protected]; Tel.: +82-31-400-5804; Fax: +82-31-400-5958

Received: 1 June 2017; Accepted: 20 July 2017; Published: 24 July 2017

Abstract: The extract of Hedera helix L. (Araliaceae), a well-known folk medicine, has been popularly used to treat respiratory problems, worldwide. It is very likely that this herbal extract is taken in combination with conventional drugs. The present study aimed to evaluate the effects of H. helix extract on (CYP) -mediated metabolism to predict the potential for herb–drug interactions. A cocktail probe assay was used to measure the inhibitory effect of CYP. H. helix extracts were incubated with pooled human liver microsomes or CYP isozymes with CYP-specific substrates, and the formation of specific metabolites was investigated to measure the inhibitory effects. H. helix showed significant inhibitory effects on CYP2C8, CYP2C19 and CYP2D6 in a concentration-dependent manner. In recombinant CYP2C8, CYP2C19 and CYP2D6 isozymes, the IC50 values of the extract were 0.08 ± 0.01, 0.58 ± 0.03 and 6.72 ± 0.22 mg/mL, respectively. Further investigation showed that H. helix extract has a positive time-dependent inhibition property on both CYP2C8 and CYP2C19 with IC50 shift value of 2.77 ± 0.12 and 6.31 ± 0.25, respectively. Based on this in vitro investigation, consumption of herbal medicines or dietary supplements containing H. helix extracts requires careful attention to avoid any CYP-based interactions.

Keywords: Hedera helix; Araliaceae; CYP inhibition; human liver microsomes; herb–

1. Introduction Hedera helix L. (Araliaceae), also known as common ivy or English ivy, has been traditionally used for the treatment of respiratory disorders [1]. The pharmacological data of H. helix extracts, including its bronchodilator, antibacterial, bronchospasmolytic, and expectorant effects, have supported its traditional use as a natural remedy for respiratory illness [2,3]. Currently, it is one of the top-selling herbal respiratory medicines in many countries worldwide, and it is popularly used for the treatment of cough and cough-related problems [4,5]. Bronchospasmolytic activity was exerted by hederacoside C, α-hederin, aglycone hederagenin, kaempferol and of H. helix extract [6]. Apigenin, kaempferol and quercetin significantly reduced the contraction of guinea-pig isolated ileum induced by prostaglandin E2 and leukotriene D4 [7]. The saponin from H. helix inhibited the terbutaline-stimulated internalization of the β2-adrenergic receptor in alveolar epithelial type-II cell line to explain its

Molecules 2017, 22, 1241; doi:10.3390/molecules22071241 www.mdpi.com/journal/molecules Molecules 2017, 22, 1241 2 of 10 spasmolytic and β-mimetic effects [8,9]. Hederacoside C (HDC) is known as one of the primary constituents responsible for the therapeutic efficacy of H. helix extracts [10]. Unlike conventional drugs, herbal products are a complex mixture of bioactive constituents. As a result, their co-administration with prescription drugs may produce unexpected toxic or adverse consequences [11]. The main mechanisms underlying such interactions are via pharmacokinetic modulations such as inhibition or induction of drug-metabolizing and transporters. Among them, the inhibition of cytochrome P450 (CYP), a representative drug-metabolizing enzyme, is considered as one of the most frequent causes for herb–drug interactions [11,12]. Therefore, evaluating the inhibition of CYP enzyme activity by herbal and herb-derived medicine is vital to predict any possible pharmacokinetic interactions with conventional drugs and to characterize their safety profile. Due to its properties as a respiratory remedy and a traditional herbal medicine, H. helix extracts are very likely to be used as an adjuvant to conventional drugs in treating various diseases accompanied by respiratory disorders. In this context, it is necessary to investigate and characterize the drug interactions with H. helix extracts to ensure safe use. It has been reported that liver enzymes are the major metabolizing enzymes to convert the principal bioactive constituents of H. helix to the secondary metabolites [13,14]. In two in vivo interaction studies [15,16], subcutaneously administered α-hederin influenced P450 enzymes in a dose-dependent manner, but no clinical relevance was expected from the results, as the IC50 values are high in comparison with its [14]. However, to our knowledge, no previous studies have investigated how H. helix whole extracts affect CYP enzyme activity. Here, we examined the inhibitory effects of H. helix extract (as a whole) and its major bioactive constituent HDC on CYP450-mediated drug metabolism using human liver microsomes and individual recombinant CYP isozymes.

2. Results

2.1. CYP Inhibition Assay in Pooled Human Liver Microsomes We investigated the inhibitory effect of H. helix extract on CYP enzymes in pooled human liver microsomes. The CYP inhibition assay system was confirmed with the following well-known CYP-selective inhibitors: furafylline for CYP1A2, methoxsalen for CYP2A6, quercetin for CYP2C8, sulfaphenazole for CYP2C9, for CYP2C19, quinidine for CYP2D6, and for CYP3A4. Each of these inhibitors reduced the formation of each corresponding CYP-specific metabolite by >95%, indicating that the assay system was functioning well. The activities of seven CYP isozymes were tested with various concentrations of H. helix extracts, and the amount of metabolite produced at each concentration was measured. Figure1 presents the representative multiple reaction monitoring (MRM) chromatograms of the control and H. helix extract/HDC-treated human liver microsome samples. Notably, H. helix extracts showed significant inhibitory activity against CYP2C8, CYP2C19, and CYP2D6 enzyme activity in a concentration-dependent manner (Figure2A,C). The IC50 values of the extract against CYP2C8, CYP2C19 and CYP2D6 were 0.13 ± 0.01, 1.04 ± 0.06 and 7.41 ± 0.09 mg/mL, respectively. The inhibitory effects of the extracts on the other CYP isozymes were negligible at all the concentrations tested. As HDC is a known principal bioactive component of the H. helix extract, its effects on CYPs were also investigated. The resulting data showed slight inhibition of the CYP2C8 isozyme (18%) by HDC (Figure2B,D), indicating that HDC is not primarily responsible for the CYP inhibition of H. helix extract. Molecules 2017, 22, 1241 3 of 10 Molecules 2017, 22, 1241 3 of 10 Molecules 2017, 22, 1241 3 of 10

(A) (B) (C)

(A) (B) 4 +ESI MRM Frag=90.0V [email protected](C) (152.1000 -> 110.1000) CYP_… x10 4 +ESI MRM Frag=90.0V [email protected] (152.1000 -> 110.1000) CYP_… x10 4 +ESI MRM Frag=90.0V [email protected] (152.1000 -> 110.1000) CYP_… x10 114 +ESI MRM Frag=90.0V [email protected] (152.1000 -> 110.1000) CYP_… x10 54 +ESI11 MRM Frag=90.0V [email protected] (152.1000 -> 110.1000) CYP_… x105 4 11+ESI MRM Frag=90.0V [email protected] (152.1000 -> 110.1000) CYP_… x105 11 (a) 5 115 115

(a) 0 0 0 x10 3 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… x10 03 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… x10 30 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… 0 1111113 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… x10 3 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… x10 3 +ESI MRM Frag=110.0V [email protected] (162.9000 -> 106.9000) CYP… x10 2 2 (b) 2 111111 2 2 (b) 20 0 0 2 1 x10 2 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… x10 0 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… x10 0 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… 0 11 2 111 11x10 2 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… x10 2 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… x10 +ESI MRM Frag=110.0V [email protected] (870.4000 -> 286.1000) CYP… 2 (c) 115 1111 2 2 (c) 5 4 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x10 4 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x10 4 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x10 11 11114 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x10 4 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x102 4 +ESI MRM Frag=132.0V [email protected] (312.2000 -> 230.9000) CYP… x10 2 2 (d) 111111 2 2 (d) 20 0 0 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 0 0 0 11115 11 5 1 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 3 +ESI MRM Frag=78.0V [email protected] (235.0000 -> 150.1000) CYP_… x10 (e) 11 5 111 115 (e) 0 0 0 5 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… x10 5 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… x10 4 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… x10 0 0 0 111111 5 4 x10 5 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… (f) x10 1 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… x105 +ESI MRM Frag=170.0V [email protected] (258.3800 -> 157.1000) CYP… 1 111111 (f) 10 05 0 1 3 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… x10 3 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… x10 3 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… x10 0 0 5 0 5 115 1111 3 3 x10 3 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… (g) x10 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… x10 +ESI MRM Frag=135.0V [email protected] (343.1000 -> 325.1000) CYP… 2.5 2.5 5 5 115 1111 (g) 0 0 0 2.5 2.5 4 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… x10 4 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… x10 4 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… x10 0 0 0 111111 4 4 2.5x10 4 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… (h) x102.5 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… x102.5 +ESI MRM Frag=135.0V [email protected] (472.4000 -> 436.4000) CYP… 111111 0 0 0 2.5 (h) 2.5 2.5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Counts vs. Acquisition Time (min) 0 Counts vs. Acquisition Time (min) 0 Counts vs. Acquisition Time (min) 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) Counts vs. Acquisition Time (min) FigureFigure 1. Representative 1. Representative multiple multiplereaction monitoring reaction monitoring (MRM) chromatograms (MRM) chromatograms of human liver of microsome human Figuresamplesliver 1. of, microsomeRepresentative (A) control; samples ( Bmultiple) H. helix of, reaction (Aextract-treated,) control; monitoring (B )andH. (MRM) (C helix) Hederacoside extract-treated,chromatograms C (HDC)-treated. andof human (C) Hederacoside liver A fraction microsome of sampleshumanC (HDC)-treated. liver of, (microsomalA) control; A(wasB) fraction H. incubated helix ofextract-treated, humanwith the liversubstr and microsomalate ( Cmixture,) Hederacoside wasin an incubatedNADPH-generating C (HDC)-treated. with the system,A fraction and of humanH. helixmixture, extractliver inmicrosomal (2.5 an mg/mL) NADPH-generating was or HDCincubated (100 µM) system,with for the 30 andsubstr minH. anated helix themixture, cytochromesextract in (2.5an NADPH-generating mg/mL)P450 (CYP)-specific or HDC (100metabolitesystem,µM) and H.formation helixfor 30extract was min determined(2.5 and mg/mL) the cytochromes by or LC-MS/MS.HDC (100 P450 µM) (a) Acetaminop (CYP)-specificfor 30 min anhen;d the metabolite (b) cytochromes 7-OH coumarin; formation P450 (c) (CYP)-specific was 6-OH- determined metabolite (d) by 4- formationOH-;LC-MS/MS. was determined (e) (a) 4-OH-; Acetaminophen; by LC-MS/MS. (f) (b) dextrorpha (a) 7-OH Acetaminop coumarin;n; (g)hen; 1-OH-madazolam; (c) (b) 6-OH-paclitaxel7-OH coumarin; and (h) (d)(c) terfenadine 6-OH-paclitaxel 4-OH-diclofenac; (IS). (d) 4- OH-diclofenac;(e) 4-OH-mephenytoin; (e) 4-OH-mephenytoin; (f) dextrorphan; (f) dextrorpha (g) 1-OH-madazolam;n; (g) 1-OH-madazolam; and (h) terfenadine and (h) (IS).terfenadine (IS). (A) (B) 100 (A) 100(B) 100 100 80 80

80 80 60 60

60 60 40 40

40 40 20 20 Metabolite formation(% of control) Metabolite formation (%of control)

20 20 Metabolite formation(% of control) Metabolite formation (%of control) 0 0 1A2 2A6 2C8 2C9 2C19 2D6 3A4 1A2 2A6 2C8 2C9 2C19 2D6 3A4

0 H. helix extracts (5 mg/mL) 0 HederacosideC (500 μM) 1A2 2A6 2C8 2C9 2C19 2D6 3A4 1A2 2A6 2C8 2C9 2C19 2D6 3A4 H. helix extracts (5 mg/mL) HederacosideC (500 μM) Figure 2. Cont.

Molecules 2017, 22, 1241 4 of 10 Molecules 2017, 22, 1241 4 of 10

Molecules(C) 2017, 22, 1241 (D) 4 of 10 100 100 (C) (D) 80 100 10080

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40 40 20 CYP 2C8 20 CYP 2C8 CYP 2C19 CYP 2C19 Metabolite formation (% of control) (% of formation Metabolite Metabolite formation (% of control) (% of formation Metabolite CYP 2D6 CYP 2D6 20 CYP 2C8 20 CYP 2C8 0 0 CYP 2C19 CYP 2C19 Metabolite formation (% of control) (% of formation Metabolite Metabolite formation (% of control) (% of formation Metabolite 0.01CYP 2D6 0.1 1 10 1CYP 2D6 10 100 1000 0 H. helix extract (mg/mL) 0 HederacosideC (μM) 0.01 0.1 1 10 1 10 100 1000 FigureFigure 2. Effects 2. Effects of H. of helixH. helix helix extract extractextract (mg/mL) and andhederacoside hederacoside C on C the on CYP-speci the CYP-specificfic metaboliteHederacosideC metabolite formation (μM) formation in human in liver microsomes. (A) H. helix extract (5 mg/mL); (B) Hederacoside C (500 µM); (C) Effectsµ of H. helix extract Figurehuman 2. liver Effects microsomes. of H. helix extract (A) H. and helix hederacosideextract (5 mg/mL); C on the (CYP-speciB) Hederacosidefic metabolite C (500 formationM); (C )in Effects human on theliverof H. metabolic microsomes. helix extract activities (A on) H. the helixof metabolic CYP2C8, extract (5 activitiesCYP2C19, mg/mL); of ( Band CYP2C8,) Hederacoside CYP2D6; CYP2C19, and C (500 (D and) µM);Effects CYP2D6; (C ) ofEffects hederacoside and of ( DH.) helix Effects Cextract on of the metaboliconhederacoside the metabolicactivities C on ofactivities theCYP2C8, metabolic of CYP2C19,CYP2C8, activities CYP2C19, and of CYP2D6. CYP2C8, and CYP2D6; Data CYP2C19, was and presented and (D CYP2D6.) Effects as the of Data hederacosidemean was ± presentedS.D. ofC theon as thedata obtainedmetabolicthe mean from activities± twoS.D. independent of of the CYP2C8, data obtained experiments. CYP2C19, from and two CYP2D6. independent Data was experiments. presented as the mean ± S.D. of the data obtained from two independent experiments. 2.2.2.2. CYP CYP Inhibition Inhibition Assay Assay in cDNA-Expressed in cDNA-Expressed Recombinant Recombinant CYP CYP Isozymes Isozymes 2.2. CYP Inhibition Assay in cDNA-Expressed Recombinant CYP Isozymes TheThe results results of ofthe the initial initial assessment assessment with with human liverliver microsomesmicrosomesrevealed revealed that thatH. H. helix helixextract extract significantlysignificantlyThe resultsinhibited inhibited of the the enzymeinitial enzyme assessment activities activities ofwith of all all threhuman threee enzymes. enzymes. liver microsomes Accordingly, Accordingly, revealed the the CYP that inhibitory H. helix extract effecteffect of H. helixofsignificantlyH. extract helix extract was inhibited further was the furtherinvestigated enzyme investigated activities using ofthe usingall cDNA thre thee-expressed enzymes. cDNA-expressed Accordingly, recombinant recombinant the CYP CYP isozymes inhibitory CYP isozymesfor effect CYP2C8, of CYP2C19,forH. helix CYP2C8, extractand CYP2D6. CYP2C19,was further As and ainvestigated result CYP2D6., the usinginhibitory As the a result, cDNA effect-expressed the of the inhibitory H. recombinanthelix effect extract of CYP on the CYP2C8, isozymesH. helix CYP2C19forextract CYP2C8, on and CYP2C19, and CYP2D6. As a result, the inhibitory effect of the H. helix extract on CYP2C8, CYP2C19 and CYP2D6CYP2C8, was CYP2C19concentration-dependent, and CYP2D6 was as shown concentration-dependent, in the assay with human as shownliver microsomes in the assay (Figure with 3). human The IC50 CYP2D6 was concentration-dependent, as shown in the assay with human liver microsomes (Figure 3). The IC50 valuesliver of microsomes the extract against (Figure CYP2C8,3). The IC CYP2C1950 values and of the CYP2D6 extract were against 0.08 CYP2C8, ± 0.01, 0.58 CYP2C19 ± 0.03 and and 6.72 CYP2D6 ± 0.22 weremg/mL, values of the extract against CYP2C8, CYP2C19 and CYP2D6 were 0.08 ± 0.01, 0.58 ± 0.03 and 6.72 ± 0.22 mg/mL, respectively,0.08 ± 0.01, which 0.58 ±are0.03 lower and than 6.72 the± 0.22 corresponding mg/mL, respectively, levels in human which areliver lower microsomes. than the correspondingAdditionally, the respectively, which are lower than the corresponding levels in human liver microsomes. Additionally, the inhibitorylevels in effect human of HDC liver microsomes. against recombinant Additionally, CYP theisozymes inhibitory was effect found of to HDC be negligible against recombinant (data not shown); CYP inhibitory effect of HDC against recombinant CYP isozymes was found to be negligible (data not shown); thisisozymes finding was was consistent found to bewith negligible the data (data of human not shown); liver microsomes, this finding confirming was consistent that withthe CYP the datainhibitory of this finding was consistent with the data of human liver microsomes, confirming that the CYP inhibitory effect of H. helix extract was not attributed to HDC. H. helix humaneffect of liverH. helix microsomes, extract was confirming not attributed that to the HDC. CYP inhibitory effect of extract was not attributed to HDC. (A) (B) (A) (B) 100 100 100 100

100 100 80 80 80 80 80 80

60 60 60 60 60 60

40 40 40 40 4040

20 20 Co-incubationCo-incubation 20 20 Pre-incubationPre-incubation with NGSwith NGS 2020 Co-incubation Co-incubationCo-incubationPre-incubationPre-incubation without without NGS NGS Co-incubation 6-OH paclitaxel formation (% of control) (% formation paclitaxel 6-OH 6-OH paclitaxel formation (% of control) (% formation paclitaxel 6-OH 0 Pre-incubation0 Pre-incubation with withNGS NGS Pre-incubation withwith NGS NGS Pre-incubation0.01Pre-incubation0.01 without 0.1without 0.1NGS NGS 1 1 10 10 Pre-incubation withoutwithout NGS NGS 6-OH paclitaxel formation (% of control) 6-OH paclitaxel formation (% of control) 0 H. helixH. helix extract extract (mg/mL) (mg/mL) 0 0 control) of (% formation mephenytoin 4-OH 0 4-OH mephenytoin formation (% of control) of (% formation mephenytoin 4-OH 0.010.01 0.1 0.1 1 1 10 10 0.010.01 0.1 0.1 1 1 10 10 H. helixH. helix extract extract (mg/mL) (mg/mL) H.H. helixhelix extract extract (mg/mL) (mg/mL) Figure 3. Cont.

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(C)

100

80

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20 Co-incubation Pre-incubation with NGS Pre-incubation without NGS

Dextromethorphan formation (% of control) (% of formation Dextromethorphan 0 0.01 0.1 1 10 H. helix extract (mg/mL)

FigureFigure 3. 3. CYP-speciCYP-specificfic metabolite metabolite formation formation as the percent as the of percent control after of control co-incubation after co-incubationand pre-incubation and pre-incubationof H. helix extracts of withH. helix and withoutextracts NADPH with and in c-DNA without expressed NADPH CYP isozymes; in c-DNA (A expressed) CYP2C8; (B CYP) CYP2C19, isozymes; and (C) CYP2D6. Data was presented as the mean ± S.D. of the data obtained from two independent experiments. (A) CYP2C8; (B) CYP2C19, and (C) CYP2D6. Data was presented as the mean ± S.D. of the data 2.3.obtained Time-Dependent from two Inhibition independent (TDI) experiments.and Mechanism-based Inactivation (MBI) Tests with Recombinant CYP2C8, CYP2C19 and CYP2D6 Isozymes 2.3. Time-Dependent Inhibition (TDI) and Mechanism-based Inactivation (MBI) Tests with Recombinant CYP2C8,To CYP2C19elucidate whether and CYP2D6 the inhibition Isozymes of CYP by H. helix extracts was time-dependent, the IC50 values derived from the co-incubation and pre-incubation assays were compared. Thus, the H. helix extract in recombinantTo elucidate CYP2C8, whether CYP2C19 the inhibition and CYP2D6 of CYPisozymes by H. was helix pre-incubatedextracts was for time-dependent,20 min with NADPH the and IC50 valuesthe IC derived50 value fromshift for the co-incubation co-incubation was and calculated. pre-incubation The inhibitory assays effects were of compared. H. helix extracts Thus, on the CYP2C8H. helix extractand CYP2C19 in recombinant were more CYP2C8, potent CYP2C19after pre-incubation and CYP2D6 (Figure isozymes 3), which was suggested pre-incubated that the forH. helix 20 min extract with may act as a time-dependent inhibitor of CYP2C8 and CYP2C19. The IC50 shift for CYP2C8, CYP2C19 and NADPH and the IC50 value shift for co-incubation was calculated. The inhibitory effects of H. helix extractsCYP2D6 on from CYP2C8 the co-incubation and CYP2C19 assay were to the more pre-incubation potent after assay pre-incubation was 3.01 ± 0.02, (Figure 1.88 3± ),0.01 which and 1.21 suggested ± 0.06 mg/mL, respectively. The fold shift was calculated as the ratio of the IC50 of co-incubation (IC50-co) to that of that the H. helix extract may act as a time-dependent inhibitor of CYP2C8 and CYP2C19. The IC50 the pre-incubation with NADPH IC50-pre as follows: shift for CYP2C8, CYP2C19 and CYP2D6 from the co-incubation assay to the pre-incubation assay was 3.01 ± 0.02, 1.88 ± 0.01 and 1.21 ± 0.06 mg/mL,IC50 shift-fold respectively. = IC50-co/IC The50-pre fold shift was calculated as the ratio of the ICCompounds50 of co-incubation showing an (IC IC50-co50 shift) to thatof ≥1.5 of can the be pre-incubation classified as positive with NADPH for TDI IC[17,18].50-pre Basedas follows: on this criterion, H. helix extracts may have a time-dependent inhibitory effect on CYP2C8 and CYP2C19. Another IC50 shift approach [19,20] was performedIC50 shift-fold to identify = ICthe50-co/ potentialIC50-pre of MBI. For this, the IC50 shift from the pre-incubation assay in the absence and presence of NADPH was investigated (Figure 3). As a result, the IC50Compounds shift for CYP2C8, showing CYP2C19 an IC and50 shift CYP2D6 of ≥ 1.5was can 2.77 be ± 0.12 classified, 6.31 ± as0.25, positive and 1.38 for ± TDI0.08 mg/mL, [17,18]. respectively. Based on this criterion,CompoundsH. helix showingextracts a ratio may of have IC50 ashift time-dependent of ≥4 can be classified inhibitory as positive effect on for CYP2C8 MBI [19]. and Thus, CYP2C19. H. helix extracts Another were considered to be a mechanism-based inactivator on CYP2C19. IC50 shift approach [19,20] was performed to identify the potential of MBI. For this, the IC50 shift from the pre-incubation assay in the absence and presence of NADPH was investigated (Figure3). As a result, 3. Discussion the IC50 shift for CYP2C8, CYP2C19 and CYP2D6 was 2.77 ± 0.12, 6.31 ± 0.25, and 1.38 ± 0.08 mg/mL, CYP enzymes are vital to the metabolism of many conventional and herbal medicines. Some of these respectively. Compounds showing a ratio of IC50 shift of ≥4 can be classified as positive for MBI [19]. Thus,enzymesH. helix canextracts be induced were or considered inhibited by to xenobiotics, be a mechanism-based resulting in clinically inactivator significant on CYP2C19. drug-drug or herb– drug interactions and causing unanticipated adverse reactions and/or therapeutic failure [17]. There are no 3. Discussionstandardized methods for detecting in vitro TDI, but they generally have in common a two-step process of pre-incubation with a test compound followed by the quantification of enzyme activity with a probe substrate.CYP enzymes The MBI are represents vital to the TDI, metabolism where the of inhi manybitory conventional effects are not and only herbal time-dependent medicines. but Some they of thesealso enzymes require metabolism can be induced by the or enzyme inhibited that by is xenobiotics,ultimately inactivated resulting [20]. in clinically Thus, time-dependent significant drug-drug enzyme orinhibitors herb–drug display interactions an increasing and causing degree of unanticipated enzyme inhibi adversetion with reactions an increased and/or pre-incubation therapeutic time failure with the [17 ]. Thereenzyme. are no For standardized CYP enzymes, methods a source of for NADPH detecting cofactorin vitro is oftenTDI, required but they to generallygenerate MBI. have The in enzymatic common a two-stepprocess processleading ofto pre-incubationMBI is irreversible, with and a testcatalytic compound activity followedcannot be by restored. the quantification However, the of enzyme enzyme inactivation occurring in the pre-incubation step must be distinguished from reversible inhibition by activity with a probe substrate. The MBI represents the TDI, where the inhibitory effects are not only time-dependent but they also require metabolism by the enzyme that is ultimately inactivated [20]. Thus, time-dependent enzyme inhibitors display an increasing degree of enzyme inhibition with an increased pre-incubation time with the enzyme. For CYP enzymes, a source of NADPH is often required to generate MBI. The enzymatic process leading to MBI is irreversible, and catalytic Molecules 2017, 22, 1241 6 of 10 activity cannot be restored. However, the enzyme inactivation occurring in the pre-incubation step must be distinguished from reversible inhibition by comparison to a suitable control (i.e., without NADPH) incubation, to predict NADPH-independent metabolism as well as enzyme degradation. The presence of NADPH in the absence of a substrate may accelerate enzyme activity loss (possibly due to generation of reactive oxygen species) or exert a stabilizing effect, depending on the enzyme and assay conditions [21]. Compared to reversible CYP inhibition, drug-induced MBI (a quasi-irreversible or irreversible inhibition) of CYP enzymes poses a greater safety concern because of the increased risk of pharmacokinetic interactions upon multiple dosing and the sustained duration of such interactions even after the termination of such entities [22]. Characterization of the inactivation property of CYP is essential for predictions of the drug–drug or herb–drug interaction potential of TDI-positive medicines [20]. Recently, guidelines from regulatory agencies such as the United States Food and Drug Administration [23] and pharmaceutical companies [24] have recognized the importance of mitigating drug interaction risks, particularly with respect to CYP TDI/MBI. In pre-clinical drug discovery, the in vitro assessment of PK interactions through TDI is routinely conducted for lead optimization [22]. As herbal medicines (extracts) are a complex mixture formed by various chemical entities [25], some of these constituents possibly contain the functional group responsible for the MBI of CYPs [20,26]. Many studies have reported the MBI of human CYPs by constituents isolated from traditional herbal medicines [27–29]. For the TDI assessment with H. helix extracts, IC50 shifts were calculated by comparing IC50 vales from the co-incubation assay to the pre-incubation assay [17], which showed positive results (≥1.5) for CYP2C8 and CYP2C19. As for MBI, the IC50 shift was demonstrated from the ratio of the IC50 value from the pre-incubation assay in the absence of NADPH to that from pre-incubation in its presence. Based on the resulting data, the H. helix extracts were considered positive for CYP2C19 (IC50 shift ≥4) [19]. Thus, our in vitro results indicated that H. helix shows TDI effects for CYP2C8 and CYP2C19, but it has a positive MBI response only for CYP2C19. CYP2D6 inhibition was slightly effected during this study, indicating that H. helix has minimal effects as TDI or MBI. Based on our findings, there is a potential for herb–drug interactions between the H. helix extract and commonly prescribed conventional medicines that are CYP2C8, CYP2C19 and CYP2D6 substrates. The most sensitive in vivo substrates for CYP2C8 include , and paclitaxel, while quercetin and are the inhibitors and moderate sensitive substrates for CYP2C8 [23]. , , esoprazole, and S-mephenytoin are the in vivo sensitive substrates for CYP2C19, while dextromethorphan is a substrate for CYP2D6 [23]. In particular, paclitaxel and S-mephenytoin are known to have a narrow therapeutic range, while the drugs used for respiratory illness like dextromethorphan and montelokast are quite sensitive here. Therefore, the concomitant use of these drugs with H. helix extracts may result in clinically significant drug interactions and requires careful attention. The dosage of H. helix preparations is discussed contradictorily in the literature. In a controlled study, the efficacy was shown with low dosages (approximately 300 mg herbal substance), while in the market there are preparations with daily dosages up to approximately 1000 mg herbal substance (Committee on Herbal Medicinal Products, European Medicines Agency) [4,10,14]. The clinical pharmacokinetic data on H. helix extracts is unavailable at present. Although the absorption of H. helix extract from the gastrointestinal tract is shown to be somewhat inadequate for some constituents like hederacoside C and α-hederin [10,14], if the extract is assumed as a single compound, its plasma concentration could reach up around 1 mg/mL. Therefore, it might have clinically close relevance for CYP-mediated herb–drug interactions, at least for CYP2C8 and CYP2C19, considering its maximum daily dosages.

4. Materials and Methods

4.1. Chemicals and Reagents HDC (>98.0%) and H. helix extract were obtained from the Lab. of Pharmacognosy, College of Pharmacy, Yonsei University (Incheon, Korea). The extract was prepared by extracting the pulverized Molecules 2017, 22, 1241 7 of 10 ivy leaves with 30% ethanol for 1 h using sonication. A voucher specimen of H. helix extract (HY-2016-01-05) was deposited at the Herbarium of the College of Pharmacy, Hanyang University, MoleculesAnsan, 2017 Korea., 22, 1241H. helix extract contains 8.2% of HDC. The content of HDC was determined by liquid7 of 10 chromatography–tandem mass spectrometry (LC-MS/MS) [10] and the representative chromatogram is massshown spectrometry in Figure4. (LC-MS/MS) Pooled human [10] liver and microsomes the representa andtive recombinant chromatogram CYP2C8, is shown CYP2C19, in Figure and CYP2D64. Pooled humanisozymes liver were microsomes purchased and recombinant from BD Gentest CYP2C8, (Woburn, CYP2C19, MA, and USA).CYP2D6 Glucose-6-phosphate, isozymes were purchasedβ-NADP+, from BD Gentestglucose-6-phosphate (Woburn, MA, USA). dehydrogenase, Glucose-6-phosphate, coumarin, β-NADP+, phenacetin, glucose-6-phosphate diclofenac, midazolam, dehydrogenase, mephenytoin, coumarin, phenacetin,dextromethorphan, diclofenac, ketoconazole, midazolam, mephenytoin, and terfenadine dextromethorphan, were purchased ketoconazole, from Sigma and Chemical terfenadine Co. (Saint were purchasedLouis, MO, from USA). Sigma All Chemical other solvents Co. (Saint used Louis, were MO, of HPLCUSA). gradeAll other and solvents were obtained used were from of HPLC J. T. Bakergrade and(Phillipsburg, were obtained NJ, from USA). J. DistilledT. Baker (Phillipsburg, water was prepared NJ, USA). using Distilled a Milli-Q water purification was prepared system using (Millipore, a Milli-Q purificationBillerica, MA, system USA). (Millipore, All standard Billerica, solutions MA, USA). and All mobile standard phases solutions were and passed mobile through phases were a 0.22 passedµm throughmembrane a 0.22 filter µm beforemembrane use. filter before use.

(A) (B)

1219.71 > 469.42 (Hederacoside) 469.23 100 100 2.25e3 % % 749.84 1219.71

0 m/z 0 Time 400 500 600 700 800 900 1000 1100 1200 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 Figure 4. Hederacoside C, (A) product ion mass spectra of the [M − H]− ions of HDC, and (B) MRM chromatograms Figure 4. Hederacoside C, (A) product ion mass spectra of the [M − H]− ions of HDC, and (B) MRM of HDC in H. helix extracts. chromatograms of HDC in H. helix extracts. 4.2. CYP Inhibition Assay 4.2. CYP Inhibition Assay CYP inhibition assays with human liver microsomes were performed for H. helix extract at various concentrationsCYP inhibition (0.01, 0.05, assays 0.1, with 0.5, 1.0, human 2.5 and liver 5 microsomesmg/mL) and wereHDC performed(1, 10, 100, forandH. 500 helix µM)extract according at various to the µ methodconcentrations used in (0.01,our 0.05,previous 0.1, 0.5,study 1.0, [30] 2.5 and(For 5 the mg/mL) determination and HDC of (1, the 10, IC 100,50 andvalue 500 for M)CYP2D6, according the concentrationto the method range used of in H. our helix previous extract study was extended [30] (For theup to determination 10 mg/mL). The of the reaction IC50 value mixtures for CYP2D6, consisted the of humanconcentration liver microsomes range of H. at helix a concentrationextract was extendedof 0.5 mg/mL; up to 10H. mg/mL).helix extract The or reaction HDC standard mixtures solution consisted at variousof human concentrations; liver microsomes an NADPH-generating at a concentration of system 0.5 mg/mL; (NGS,H. containing helix extract 0.1 or M HDC glucose-6-phosphate, standard solution 10at mg/mL various β concentrations;-NADP+, and glucose-6-phosphate an NADPH-generating dehydrogenase) system (NGS, at 1.0 containing U/mL; and 0.1 probe M glucose-6-phosphate, substrates (the final concentrations10 mg/mL β-NADP+, were at ~Km, and glucose-6-phosphatewhich were 40 µM ph dehydrogenase)enacetin, 2.5 µM atcoumarin, 1.0 U/mL; 5 µM and dextromethorphan, probe substrates 10(the µM final diclofenac, concentrations 160 µM mephenytoin, were at ~Km, 10 which µM paclitaxel, were 40 andµM 2.5 phenacetin, µM midazolam) 2.5 µM in coumarin, 200 µL of 50.1µ MM potassiumdextromethorphan, phosphate 10 bufferµM diclofenac,(pH 7.4). The 160 reµactionM mephenytoin, mixture was 10 pre-incubatedµM paclitaxel, at and37 °C 2.5 forµM 5 min midazolam) without NGSin 200 andµ Lthen of 0.1further M potassium incubated phosphatefor 30 min bufferwith NGS (pH in 7.4). a water The bath reaction. Ketoconazole mixture was (5 µM), pre-incubated furafylline (10at 37µM),◦C methoxsalen for 5 min without (10 µM), NGS sulfaphenazole and then further (50 µM), incubated ticlopidine for 30 (20 min µM), with quercetin NGS in a(30 water µM), bath. and quinidineKetoconazole (50 µM), (5 µ whichM), furafylline are all CYP (10 specificµM), inhibitors, methoxsalen were (10 testedµM), as sulfaphenazole positive controls (50 to confirmµM), ticlopidine substrate selectivity(20 µM), quercetinfor various (30 CYPµM), isoforms. and quinidine After the (50 incubation,µM), which the are reaction all CYP was specific stopped inhibitors, by adding were 400 tested µL of 0.1% acetic acid containing internal standard (0.16 µM terfenadine). For the further investigation of specific as positive controls to confirm substrate selectivity for various CYP isoforms. After the incubation, CYP isozyme inhibition, 12.5 pmol isozyme (CYP 2C8, 2C19, and 2D6) were used instead of human liver the reaction was stopped by adding 400 µL of 0.1% acetic acid containing internal standard (0.16 µM microsomes, and the corresponding specific substrates were added to the reaction mixture (10 µM paclitaxel, terfenadine). For the further investigation of specific CYP isozyme inhibition, 12.5 pmol isozyme (CYP 160 µM mephenytoin, and 5 µM dextromethorphan, respectively), following the procedure as described 2C8, 2C19, and 2D6) were used instead of human liver microsomes, and the corresponding specific before. For time-dependent inhibition, the reaction mixtures consisting of recombinant CYP2C8/CYP2C19/ µ µ µ CYP2D6,substrates and were NGS added in a 0.1 to M the potassium reaction ph mixtureosphate (10buffer,M was paclitaxel, pre-incubated 160 M with mephenytoin, H. helix extract and for 5 zeroM (co-incubation)dextromethorphan, and 20 respectively), min, followed following by the theaddition procedure of the as corresponding described before. substrates For time-dependent (i.e., paclitaxel (CYP2C8)inhibition, or the mephenytoin reaction mixtures (CYP2C19) consisting or dextromethorphan of recombinant (CYP2D6)), CYP2C8/CYP2C19/CYP2D6, and further incubated and for NGS 30 min in ina 0.1water M potassiumbath (followed phosphate by rest buffer,of the procedur was pre-incubatede, as described). with H.For helix the extractdetermination for zero of (co-incubation) IC50 values for mechanism-basedand 20 min, followed inactivation, by the the addition recombinant of the CYP corresponding isozyme was substrates pre-incubated (i.e., for paclitaxel 30 min in (CYP2C8) the presence or andmephenytoin absence of NGS (CYP2C19) [18]. or dextromethorphan (CYP2D6)), and further incubated for 30 min in water bath (followed by rest of the procedure, as described). For the determination of IC50 values for 4.3. Sample Preparation For sample preparation, the incubation mixtures were passed through activated Sep-Pak C18 cartridges (96-well OASIS HLB Extraction Cartridge, Waters, Milford, MA, USA). The cartridges were first eluted with

Molecules 2017, 22, 1241 8 of 10 mechanism-based inactivation, the recombinant CYP isozyme was pre-incubated for 30 min in the presence and absence of NGS [18].

4.3. Sample Preparation For sample preparation, the incubation mixtures were passed through activated Sep-Pak C18 cartridges (96-well OASIS HLB Extraction Cartridge, Waters, Milford, MA, USA). The cartridges were first eluted with methanol (1 mL) and 0.1% acetic acid (1 mL). After loading the sample, the cartridges were washed twice with 0.1% acetic acid (2 mL) and finally eluted with 1 mL of methanol. The eluate was evaporated under nitrogen gas, and the residue was reconstituted in the mobile phase (100 µL, 0.1% formic acid in an 85/15 mixture of water/acetonitrile, v/v). A 5 µL aliquot was injected into the LC-MS/MS system.

4.4. LC-MS/MS Analysis The LC-MS/MS system consisted of an Agilent 1260 series binary pump HPLC system and an Agilent 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) which was equipped with the source of an electrospray ionization (ESI). Chromatographic separation was conducted by using a Fortis-C8 column (2.1 mm × 100 mm, 5 µm; Fortis Technologies Ltd., Cheshire, UK), and the column temperature was maintained at 40 ◦C. The composition of the HPLC mobile phases were (A) 0.1% formic acid in distilled water and (B) 90% acetonitrile in the solvent A. A gradient program was used at a flow rate of 0.2 mL/min; the composition of solvent B was initially set at 15%, then gradually increased to 85% (15−85%) over 3 min, and maintained for 1.5 min (85%), and finally re-equilibrium for 3.5 min (85−15%). The mass spectrometer was operated in the positive ion mode, with MRM. The precursor–product ion pairs (Q1/Q3) used in MRM mode are shown in Table1.

Table 1. Information on the probe substrates and their corresponding CYP-specific metabolites used in this study.

PROBE Substrate Metabolite Precursor-Ion Daughter-Ion P450 Isozyme Substrate Conc. (µM) Monitored (m/z) (m/z) CYP1A2 Phenacetin 40 Acetaminophen 152.1 110.1 CYP2A6 Coumarin 2.5 7-OH-coumarin 162.9 106.9 CYP2C8 Paclitaxel 10 6-OH-paclitaxel 870.4 286.1 CYP2C9 Diclofenac 10 4-OH-diclofenac 312.2 230.9 CYP2C19 Mephenytoin 160 4-OH-mephenytoin 235.0 150.1 CYP2D6 Dextromethorphan 5 Dextrorphan 258.3 157.1 CYP3A4 Midazolam 2.5 1-OH-midazolam 343.1 325.1 Internal Standard - Terfenadine 472.4 436.4

4.5. Statistics Data was presented as the mean ± S.D. of the data obtained from two independent experiments. The IC50 value was calculated with the metabolite-formation values based on the four parameters logistic regression using Sigmaplot v12.5 (Systat Software, Inc., WPCubed GmbH, Munich, Germany) and GraphPad Prism v7.1 (GraphPad Software, Inc., La Jolla, CA, USA).

5. Conclusions The H. helix extract is a popularly used traditional medicine as well as one of the top-selling herbal medicines for respiratory disorders in many countries. Despite its high possibility of co-administration with other prescribed drugs, no studies have investigated its inhibitory potential on CYP enzyme activities. In the present study, we reported CYP enzyme inhibition by H. helix extracts for the first time. H. helix extract inhibited the enzyme activities of CYP2C8, CYP2C19 and CYP2D6 in a concentration-dependent manner, while it showed time-dependent inhibition for CYP2C8 and CYP2C19. Furthermore, our results suggested that H. helix reacts as a mechanism-based inactivator for Molecules 2017, 22, 1241 9 of 10

CYP2C19 (IC50 shift ≥4). Based on these findings, consuming herbal medicines or dietary supplements containing H. helix extracts requires careful attention and further in vivo studies should investigate CYP-mediated interactions with H. helix to verify the potential of herb–drug interactions and to establish proper recommendations for the safe therapeutic use of H. helix.

Acknowledgments: This work was supported by the research grant through the National Research Foundation of Korea (NRF-2016K1A3A1A20005958). Author Contributions: H.H.Y., S.H.K. and Y.Z. conceived and designed the experiments; S.U.R. performed the experiments; M.S.C. and Y.Z. analyzed the data; I.S.K. and M.S.C. contributed analysis tools; S.U.R. and H.H.Y. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Not available.

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